Effectiveness of feeding different biochars on growth, digestibility, body composition, hematology and mineral status of the Nile tilapia, Oreochromis niloticus

Oreochromis niloticus fingerlings (5.15 ± 0.02 g; n = 315) were fed with different types of biochar (BC)-supplemented sunflower meal-based (SFM) diet to investigate the effects of various BC inclusions on their nutritional digestibility, body composition, hematology and mineral status for 60 days. Seven different diets were formulated based on the SFM based diet: one was a control (TD-I, CON) and the other six diets were supplemented with 2% BC derived from different sources. These BCs were derived from the following: cotton stick (CSBC, TD-II), wheat straw (WSBC, TD-III), corn cob (CCBC, TD-IV), house waste (HWBC, TD-V), grass waste (GWBC, TD-VI), and green waste (GwBC, TD-VII) biochar. There were three replicates for each test diet. Each tank had fifteen tilapia fingerlings, and they were fed with 5% of their live wet weight and twice daily. The outcomes showed that the supplementation of CCBC significantly elevated the growth, nutrient absorption, and body composition of the O. niloticus fingerlings (p < 0.05); with concomitant lowering of the quantity of nutrients released into the water bodies whereas HWBC gave negative impacts. The maximal mineral absorption efficiency (Ca, Na, K, Cu, Fe, P, and Zn) was achieved by the supplementation of 2% CCBC. All hematological parameters showed positive improvements (p < 0.05) with CCBC. Interestingly, CCBC significantly improved the growth, digestibility, body composition, hematology, and mineral status of O. niloticus.

Fishes, being at the tertiary level of the food chain, provide humans with a crucial and abundant source of nutrition.The proteins, minerals, unsaturated fats, and vitamins present in fish make it an excellent dietary choice 1 .The American Heart Association stated that eating fish twice a week is enough to fulfill one's daily needs for omega-3 fatty acids 2 .
The most significant input and a limiting component in aquaculture is the availability of feed.Commercial aqua-feeds are usually a mixture of several feed ingredients that provide the cultivated aquatic organisms with the essential nutrients that they need.Owing to their advantageous qualities, fishmeal (FM) and fish oil (FO) are regarded as the most important feed components 3 .High-quality animal protein is primarily supplied by aquaculture, which also makes a crucial role in global food safety.Owing to the increase in population, it is expected to grow further in response to rising demand 4 .More than 70% of the world's aquaculture food production comes from freshwater aquaculture 5 .In most countries, it has become an important industry due to high demand of fish

Experimental trial
We purchased fingerlings of O. niloticus from Fish Seed Hatchery, Faisalabad, Pakistan.The average size of the total 315 fishes was 5.15 ± 0.02 g.The fingerlings were brought to the lab in polythene bags with adequate aeration.After being carefully transported to circular cement containers with a capacity of 400 L, they were left undisturbed for the whole night.For 10 days at 28 °C, the fingerlings were acclimatized in an aerated environment 37 .In order to avoid detrimental inflammation or ecto-parasites, the fingerlings were immersed in a NaCl solution 38 .Commercial fish feed was given to these fingerlings twice a day.

Production of biochar
Following crushing and drying, a variety of biomass materials were collected for the purpose of producing BC.These materials included cotton sticks, wheat straw, corncob, House waster, grass waste, and green waste.Separate batches of BC were pyrolyzed in a top-lit draft gasifier.After that, they were cooled and then ground into extremely fine particles by passing them through a 2 mm sieve 39 .Before being utilized to make the experimental diets, the powdered BC was stored in an airtight container.

Feed Ingredients and processing
One control diet and six experimental treatments were formulated.The test diets were supplemented with a 2% BC supplement from different sources (TD-II, corn stick BC, CSBC); (TD-III, wheat straw BC, WSBC); (TD-IV, corn cob BC, CCBC); (TD-V, house waste BC, HWBC); (TD-VI, grass waste BC, GWBC); and (TD-VII, green waste BC, GwBC), whereas the control group (TD-I, CON) did not get any BC.Once all the materials had been crushed through a 0.5 mm sieve, they were well mixed for five minutes and fish oil was gradually added.Afterwards, the components were combined with a precise amount of water (10-15% w/w) to make a homogeneous dough 40 .The dough was finally run through a pelleting machine to turn into pellets.Pellets were stored

Chemical analysis of feed, feces, and muscle
A mortar and pestle were used to homogenize samples (1 g), taken from each tank, of the test diet and feces.The materials were evaluated by standard procedures 42,43 .For 12 h, the samples were dried in an oven at 105 °C to determine its moisture content.The petroleum ether extraction technique was utilized to extract crude fat (CF) utilizing a Soxtec HT2 1045 system.The micro Kjeldahl apparatus was utilized to estimate crude protein (CP; N*6.25), and an electric furnace lit to 650 °C for 12 h was employed to identify ash.The gross energy (GE) was measured using the oxygen bomb calorimeter.In order to generate calibrated standards for mineral estimation commercial procedures were used 44 .

Nutrient digestibility
One-gram samples of dried feed, body and feces were pulverized and homogenized using a mortar and pestle for the determination of nutritional digestibility.The Standard technique by Brown 45 was used to estimate the apparent nutritional digestibility of each meal.

Hematological study
Following the 60-day feeding trial, three fish from each tank were dipped into 150 mg −1 tricane methane sulfonate for anesthesia 46 .After that, blood sample was drawn from the caudal vein by using heparinized syringe.The.A micro-hematocrit and a capillary tube were utilized to calculate the hematocrit.Platelets (PLTs), white blood cells (WBC) and red blood cells (RBCs), were carried out using an approved Neuber counting chamber 47 .
Utilizing the technique established the hemoglobin level was determined 48 .The mean hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) were determined using the formulae 49 .

Statistical analysis
The growth parameters, blood profile, whole body composition, nutrient digestibility and mineral status data were evaluated using a one-way ANOVA 50 .The difference between means was examined using Tukey's Honest Significant Difference Test; a p-value of less than 0.05 was considered significant 51 .Data analysis was performed using the CoStat Computer Package (version 6.303, USA).www.nature.com/scientificreports/for FCR and SGR (Fig. 2), with FCR of 1.51 and SGR of 1.84, respectively.The HWBC-based diet, however, had a negative effect (FCR: 2.75 SGR: 0.84).

Nutrient digestibility
According to the current investigation, a CCBC-based diet resulted in the highest significant values of nutrient digestibility (Fig. 3) in feces (crude protein: 11.07%, crude fat: 3.76%, and gross energy: 1.38%) where as HWBCbased diet (crude protein: 22.28%, crude fat: 4.62%, gross energy: 3.16%) was the only test diet that demonstrated negative results in terms of nutrient digestibility of feces.These values showed that maximal amount of nutrients has been released in the feces.The highest results of ADC% (crude protein: 70.2%, gross energy: 68.02% and crude fat: 65.34%) of O. niloticus were observed on a CCBC-based diet.The lowest results of ADC% were seen in the HWBC-based diet (crude protein: 40.15%, gross energy: 27.27% and crude fat: 56.34%).

Hematology
The hematological results are displayed in Table 3

Mineralization
The mineral status of each test diet differed markedly from that of the other (Table 4).The mineral digestibility data also revealed that the CCBC (TD-IV) had high digestibility coefficient values when compared to the other test diets.Diet TD-IV showed the best digestion values for Ca (56.81%), Na (52.61%),K (61.07%),P (66.32%),Fe (68.87%),Cu (66.21%), and Zn (68.92%) when given to O. niloticus fingerlings whereas lowest values of all these minerals were observed by HWBC (TD-V).

Discussion
Identifying the ideal feed formulation is a primary need in aquaculture.Animal based diets are considered the best sources of protein like FM but with the passage of time, prices of FM or animal based diets are increasing.The benefits of using plant based diet as a dietary additive to enhance overall performance of tilapia (O.niloticus), have drawn more attention in recent years.This research evaluated the effects of using BC derived from various sources, on growth, nutrient absorption, carcass composition, hematology and mineral status of O. niloticus.Some earlier research have reported beneficial findings of adding BC supplements in several types of livestock, including goats, pigs, poultry, and cattle [52][53][54] .Interestingly, substituting 2% CCBC in SFM-based diet yielded the greatest growth indices in tilapia.In another study, the growth parameters of O. niloticus were significantly improved after 8 weeks of supplementation Table 3. Hematology of O. niloticus fed with 2% BC supplementation.RBC = Red Blood Cell, MCH = Mean corpuscular hemoglobin, WBC = White blood cell, MCHC = Mean corpuscular hemoglobin concentration, PCV = Packed cell volume, PLT = Platelet, MCV = Mean corpuscular volume, Hb = hemoglobin concentration, Biochar (BC), Control (CON, TD-I), cotton stick BC (CSBC, TD-II), wheat straw (WSBC, TD-III), corn cob (CCBC, TD-IV), house waste (HWBC, TD-V), grass waste (GWBC, TD-VI), and green waste (GwBC, TD-VII).a-g Means of columns with different superscripts possess a significant difference (p < 0.05) and the data comprises the means of 3 replicas.I), cotton stick BC (CSBC, TD-II), wheat straw (WSBC, TD-III), corn cob (CCBC, TD-IV), house waste (HWBC, TD-V), grass waste (GWBC, TD-VI), and green waste (GwBC, TD-VII).a-g Means of columns with different superscripts possess a significant difference (p < 0.05) and the data comprises the means of 3 replicas.www.nature.com/scientificreports/ with activated charcoal at a rate of 7.0 g/kg 55 .The incorporation of activated charcoal into aquafeeds has been shown to adsorb and remove gases and contaminants from the gastrointestinal tract, thereby optimizing nutrient absorption and utilization, which is a key factor contributing to improved fish growth and feed efficiency.Likewise, with the results for Pangasius hypophthamus 56 , our study indicated the same conclusion.In this research, bamboo charcoal (2%) when fed to P. hypophthamus showed an increased growth performance.In a study conducted by Najmudeen et al. 57 , it was shown that O. mossambicus showed a notable increase in both weight and length when given Eichhornia crassipes BC at concentrations of 0.5% and 1%.Maximum growth rate was observed in fingerlings fed 1% BC.Moreover, improvements in body weight, FCR, and survival rate were noted in Plotosus lineatus (catfish) and Salmo trutta (trout) when 2% bamboo BC and 0.2/kg were added to their diets, respectively 56 .Furthermore, it was reported by Michael et al. 58 that substitution of 3% commercial wood charcoal increased the growth performance (WG:11 g, FCR: 1.07, SGR: 3.89 and PER: 2.89) of red tilapia juveniles.Current research revealed that except TD-V supplementation of 2% different types of BCs improved the nutrient digestibility of O. niloticus.It has been shown that adding BC to cattle feed improves the animals' production, health, and efficiency of nutrient intake 59 .According to Khalid et al. 60 , the addition of 2 mg/kg of poultry waste BC improved the digestion of CP (75.92%),CF (81.90%), and GE (74.84%) in Catla catla.Schubert et al. 53 used two different kinds of BC in accordance with the current research and reported that 2% BC improved the CP, CF, and GE of pigs in addition to having a positive impact on nutrient digestibility.Furthermore, Thu et al. 61 reported that feeding 4% bamboo charcoal to Japanese flounder resulted in a remarkable increase in body composition (CP: 17.5%, CF: 4.1%, moisture: 72% and ash: 3.5%) and protein digestibility, reaching 89%.
The best results for CP, CF, ash, and moisture in terms of body composition were obtained in the current research with CCBC.Yoo et al. 62 found that the combination of wood vinegar and charcoal positively correlated with carcass of Paralichthys olivaceus, the olive flounder.Thu et al. 61 found that P. olivaceus carcass significantly improved with the use of bamboo charcoal because there was a reduction in ammonia excretion, which raised the quality and protein content of the fish body.
Through the hematological observations, this study concluded that each type of BC supplementation impacted positively on fingerlings except for HWBC (TD-V).The best values of hematological parameters were noted in CCBC (TD-IV).There is little research about the effects of dietary BC on fish hematological indices; conversely, some observations have been reported in cattle and poultry farming.According to Mabe et al. 63 , Cyprinus carpio when fed bamboo BC showed no changes in growth indices; however, their serum quality had improved, suggesting better overall fish health.Elghalid 64 found that hematological traits such as Hb, RBCs and hemocytosis percentage (HCT%) improved when chicks were given diets containing 0%, 1%, 2%, 4%, 6%, or 8% biochar.According to Dim et al. 65 , when BC was given to Meleagris gallopavo (turkey) at 5 g kg −1 , 15 g kg −1 and 25 g kg −1 , 15 g kg −1 , showed significant improvements in RBC, HCT, Hb, and WBC.BC supplementation in red tilapia plausibly delivered a detoxifying effect that improved hematological parameters and decreased oxidative stress by reducing the absorption of toxins and other potentially harmful substances into the fish gut 58 .
The current study analysis of the mineral content of body revealed that the TD-IV (CCBC) diet gave the maximum quantity of minerals.When O. niloticus fingerlings were fed with the CCBC diet, the best digestibility values of Ca (62.78%), Na (57.03%),K (66.02%),P (67.97%),Fe (70.25%),Cu (67.90%), and Zn (68.92%) were achieved.Biochar has the potential to increase the levels of minerals because of its high cation-exchange capacity, which makes some minerals more readily available 66 .

Conclusion
In this current study, it was determined that 2% of CCBC delivered positively on growth, carcass, digestibility, hematology and mineralization of O. niloticus, except for HWBC (TD-V).Thus, BC can be used as an economical and environmentally sustainable supplement for fish health.

Figure 1 Figure 1 .
Figure 1 showed the growth parameters of O. niloticus fed diets supplemented with 2% of various types of BC.The size of fingerlings was observed to be comparable in the current investigation.When compared to other BCs and the control diet, fish that were given a CCBC SFM-based diet (test diet IV) exhibited a highest increase in weight gain.Test diet V, which consisted of HWBC SFM-based diet, resulted in the lowest growth rate of the fingerlings compared to the other diets, indicating detrimental effect on fingerlings growth.Even the HWBCbased diet surpassed the control diet in terms of growth.The greatest weight gains (13.52 g) and weight gain percentage (261.53%)were recorded in O. niloticus fingerlings that were fed CCBC supplemented SFM-based diet.The supplementation of HWBC-SFM based diet resulted in detrimental effect on weight gain.The results indicated an increase in growth with the exception of HWBC compared to all other diets, including the control diet.The CCBC-based diet exhibited the optimal values

Figure 4 Figure 2 .Figure 3 .
Figure4showed the carcass composition of O. niloticus fingerlings fed diets supplemented with 2% of the various BC types.The impact of different types of BCs on carcass outcomes (protein, fat, ash, and moisture) were significant (p < 0.05).The proportion of fat (6.07%) and moisture (75.10%) were lower, while the values of protein

Table 2 .
The chemical makeup (%) of the various feed components and experimental diets.